Inflammation and oxidative stress markers in type 2 diabetes patients with Advanced Carotid atherosclerosis.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a major global health issue and a significant risk factor for atherosclerosis. Atherosclerosis in T2DM patients has been associated with inflammation, insulin resistance, hyperglycemia, dyslipidemia, and oxidative stress. Identifying molecular features of atherosclerotic plaques in T2DM patients could provide valuable insights into the pathogenesis of the disease. METHODS: The MASCADI (Arachidonic Acid Metabolism in Carotid Stenosis Plaque in Diabetic Patients) study aimed to investigate the increase of 2-arachidonoyl-lysophatidylcholine (2-AA-LPC) in carotid plaques from T2DM and control patients and to explore its association with plaque vulnerability as well as with blood and intra-plaque biomarkers altered during diabetes. RESULTS: In a population of elderly, polymedicated patients with advanced stage of atherosclerosis, we found that T2DM patients had higher systemic inflammation markers, such as high-sensitivity C-reactive protein (hsCRP) and IL-1ß, higher levels of oxysterols, increased triglyceride levels, and decreased HDL levels as compared to control patients. Furthermore, 2-AA-LPC was significantly enriched in plaques from diabetic patients, suggesting its potential role in diabetic atherosclerosis. Interestingly, 2-AA-LPC was not associated with systemic markers related to diabetes, such as hsCRP, triglycerides, or HDL cholesterol. However, it was significantly correlated with the levels of inflammatory markers within the plaques such as lysophospholipids and 25-hydroxycholesterol, strengthening the link between local inflammation, arachidonic acid metabolism and diabetes. CONCLUSION: Our study is in line with a key role for inflammation in the pathogenesis of diabetic atherosclerosis and highlights the involvement of 2-AA-LPC. Further research is needed to better understand the local processes involved in the alteration of plaque composition in T2DM and to identify potential therapeutic targets. TRIAL REGISTRATION: The MASCADI was registered on ClinicalTrials.gov (clinical registration number: NCT03202823).

Profiling of lipid mediators in atherosclerotic carotid plaques from type 2 diabetic and non-diabetic patients.

BACKGROUND AND AIMS: Diabetes is associated with an accelerated development of atherosclerosis. Specific mechanisms related to diabetes and hyperglycemia may play a role in this process. In particular, alterations of arachidonic acid (AA) metabolism have been reported. Our main goal was to investigate for differences in the concentration of LTB4 and RvD1 as well as selected cyclooxygenase-derived mediators in carotid plaques from diabetic and non-diabetic patients. We also aimed to analyze the relationship between omega 6 and omega 3 Poly-Unsaturated Fatty acids (PUFAs) content in the plaques and the concentrations of these lipid mediators. METHODS: 29 type 2 diabetic patients and 30 control patients admitted for surgical treatment of carotid stenosis were enrolled in the present study. Carotid plaques were harvested for in-depth lipidomic profiling. RESULTS: No differences for LTB4 or other lipid mediators were observed between diabetic and non-diabetic patients. RvD1 levels were below the threshold of quantification in most of the samples. A significant correlation was found between LTB4 and 5(S)-HETE levels. Omega 3 enrichment was not significantly different between control and diabetic plaques. There was a negative correlation between DHA/AA ratio and the level of 5(S)-HETE while there was a positive association with TXB2 and PGD2 concentrations. CONCLUSION-PERSPECTIVES: Our results does not support the hypothesis of a specific involvement of LTB4 or COX-derived mediators in diabetic atherosclerosis. The relationship between DHA enrichment and the concentrations of specific inflammatory mediators within the plaque is of interest and will need to be confirmed in larger studies.

Deletion of lysophosphatidylcholine acyltransferase 3 in myeloid cells worsens hepatic steatosis after a high-fat diet.

Recent studies have highlighted an important role for lysophosphatidylcholine acyltransferase 3 (LPCAT3) in controlling the PUFA composition of cell membranes in the liver and intestine. In these organs, LPCAT3 critically supports cell-membrane-associated processes such as lipid absorption or lipoprotein secretion. However, the role of LPCAT3 in macrophages remains controversial. Here, we investigated LPCAT3's role in macrophages both in vitro and in vivo in mice with atherosclerosis and obesity. To accomplish this, we used the LysMCre strategy to develop a mouse model with conditional Lpcat3 deficiency in myeloid cells (Lpcat3KOMac). We observed that partial Lpcat3 deficiency (approximately 75% reduction) in macrophages alters the PUFA composition of all phospholipid (PL) subclasses, including phosphatidylinositols and phosphatidylserines. A reduced incorporation of C20 PUFAs (mainly arachidonic acid [AA]) into PLs was associated with a redistribution of these FAs toward other cellular lipids such as cholesteryl esters. Lpcat3 deficiency had no obvious impact on macrophage inflammatory response or endoplasmic reticulum (ER) stress; however, Lpcat3KOMac macrophages exhibited a reduction in cholesterol efflux in vitro. In vivo, myeloid Lpcat3 deficiency did not affect atherosclerosis development in LDL receptor deficient mouse (Ldlr-/-) mice. Lpcat3KOMac mice on a high-fat diet displayed a mild increase in hepatic steatosis associated with alterations in several liver metabolic pathways and in liver eicosanoid composition. We conclude that alterations in AA metabolism along with myeloid Lpcat3 deficiency may secondarily affect AA homeostasis in the whole liver, leading to metabolic disorders and triglyceride accumulation.

Regulation of glycolytic genes in human macrophages by oxysterols: a potential role for liver X receptors.

BACKGROUND AND PURPOSE: Subset of macrophages within the atheroma plaque displays a high glucose uptake activity. Nevertheless, the molecular mechanisms and the pathophysiological significance of this high glucose need remain unclear. While the role for hypoxia and hypoxia inducible factor 1α has been demonstrated, the contribution of lipid micro-environment and more specifically oxysterols is yet to be explored. EXPERIMENTAL APPROACH: Human macrophages were conditioned in the presence of homogenates from human carotid plaques, and expression of genes involved in glucose metabolism was quantified. Correlative analyses between gene expression and the oxysterol composition of plaques were performed. KEY RESULTS: Conditioning of human macrophages by plaque homogenates induces expression of several genes involved in glucose uptake and glycolysis including glucose transporter 1 (SLC2A1) and hexokinases 2 and 3 (HK2 and HK3). This activation is significantly correlated to the oxysterol content of the plaque samples and is associated with a significant increase in the glycolytic activity of the cells. Pharmacological inverse agonist of the oxysterol receptor liver X receptor (LXR) partially reverses the induction of glycolysis genes without affecting macrophage glycolytic activity. Chromatin immunoprecipitation analysis confirms the implication of LXR in the regulation of SLC2A1 and HK2 genes. CONCLUSION AND IMPLICATIONS: While our work supports the role of oxysterols and the LXR in the modulation of macrophage metabolism in atheroma plaques, it also highlights some LXR-independent effects of plaques samples. Finally, this study identifies hexokinase 3 as a promising target in the context of atherosclerosis. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

Interplay between Liver X Receptor and Hypoxia Inducible Factor 1α Potentiates Interleukin-1ß Production in Human Macrophages.

Low-grade inflammation is constitutive of atherosclerosis, and anti-inflammatory therapy inhibiting interleukin-1ß (IL-1ß) reduces the rate of cardiovascular events. While cholesterol accumulation in atheroma plaque and macrophages is a major driver of the inflammatory process, the role of the LXR cholesterol sensors remains to be clarified. Murine and human macrophages were treated with LXR agonists for 48 h before Toll-like receptor (TLR) stimulation. Unexpectedly, we observe that, among other cytokines, LXR agonists selectively increase IL1B mRNA levels independently of TLR activation. This effect, restricted to human macrophages, is mediated by activation of HIF-1α through LXR. Accordingly, LXR agonists also potentiate other HIF-1α-dependent pathways, such as glycolysis. Treatment of human macrophages with carotid plaque homogenates also leads to induction of IL1B in an LXR-dependent manner. Thus, our work discloses a mechanism by which cholesterol and oxysterols trigger inflammation in atherosclerosis. This suggests perspectives to target IL-1ß production in atherosclerotic patients.

Macrophage fatty acid metabolism and atherosclerosis: The rise of PUFAs.

Among the pathways involved in the regulation of macrophage functions, the metabolism of unsaturated fatty acids is central. Indeed, unsaturated fatty acids act as precursors of bioactive molecules such as prostaglandins, leukotrienes, resolvins and related compounds. As components of phospholipids, they have a pivotal role in cell biology by regulating membrane fluidity and membrane-associated cellular processes. Finally, polyunsaturated fatty acids (PUFAs) are also endowed with ligand properties for numerous membrane or nuclear receptors. Although myeloid cells are dependent on the metabolic context for the uptake of essential FAs, recent studies showed that these cells autonomously handle the synthesis of n-3 and n-6 long chain PUFAs such as arachidonic acid and eicosapentaenoic acid. Moreover, targeting PUFA metabolism in macrophages influences pathological processes, including atherosclerosis, by modulating macrophage functions. Omics evidence also supports a role for macrophage PUFA metabolism in the development of cardiometabolic diseases in humans. Currently, there is a renewed interest in the role of n-3/n-6 PUFAs and their oxygenated derivatives in the onset of atherosclerosis and plaque rupture. Purified n-3 FA supplementation appears as a potential strategy in the treatment and prevention of cardiovascular diseases. In this context, the ability of immune cells to handle and to synthesize very long chain PUFA must absolutely be integrated and better understood.

Revisiting the Role of LXRs in PUFA Metabolism and Phospholipid Homeostasis.

Liver X receptors (LXRs) play a pivotal role in fatty acid (FA) metabolism. So far, the lipogenic consequences of in vivo LXR activation, as characterized by a major hepatic steatosis, has constituted a limitation to the clinical development of pharmacological LXR agonists. However, recent studies provided a different perspective. Beyond the quantitative accumulation of FA, it appears that LXRs induce qualitative changes in the FA profile and in the distribution of FAs among cellular lipid species. Thus, LXRs activate the production of polyunsaturated fatty acids (PUFAs) and their distribution into phospholipids via the control of FA desaturases, FA elongases, lysophosphatidylcholine acyltransferase (LPCAT3), and phospholipid transfer protein (PLTP). Therefore, LXRs control, in a dynamic manner, the PUFA composition and the physicochemical properties of cell membranes as well as the release of PUFA-derived lipid mediators. Recent studies suggest that modulation of PUFA and phospholipid metabolism by LXRs are involved in the control of lipogenesis and lipoprotein secretion by the liver. In myeloid cells, the interplay between LXR and PUFA metabolism affects the inflammatory response. Revisiting the complex role of LXRs in FA metabolism may open new opportunities for the development of LXR modulators in the field of cardiometabolic diseases.

LPCAT3 deficiency in hematopoietic cells alters cholesterol and phospholipid homeostasis and promotes atherosclerosis.

BACKGROUND AND AIMS: LPCAT3 plays a major role in phospholipid metabolism in the liver and intestine. However, the impact of LPCAT3 on hematopoietic cell and macrophage functions has yet to be described. Our aim was to understand the functions of LPCAT3 in macrophages and to investigate whether LPCAT3 deficiency in hematopoietic cells may affect atherosclerosis development. METHODS: Mice with constitutive Lpcat3 deficiency (Lpcat3-/-) were generated. We used fetal hematopoietic liver cells to generate WT and Lpcat3-/- macrophages in vitro and to perform hematopoietic cell transplantation in recipient Ldlr-/- mice. RESULTS: Lpcat3-deficient macrophages displayed major reductions in the arachidonate content of phosphatidylcholines, phosphatidylethanolamines and, unexpectedly, plasmalogens. These changes were associated with altered cholesterol homeostasis, including an increase in the ratio of free to esterified cholesterol and a reduction in cholesterol efflux in Lpcat3-/- macrophages. This correlated with the inhibition of some LXR-regulated pathways, related to altered cellular availability of the arachidonic acid. Indeed, LPCAT3 deficiency was associated with decreased Abca1, Abcg1 and ApoE mRNA levels in fetal liver cells derived macrophages. In vivo, these changes translated into a significant increase in atherosclerotic lesions in Ldlr-/- mice with hematopoietic LPCAT3 deficiency. CONCLUSIONS: This study identifies LPCAT3 as a key factor in the control of phospholipid homeostasis and arachidonate availability in myeloid cells and underlines a new role for LPCAT3 in plasmalogen metabolism. Moreover, our work strengthens the link between phospholipid and sterol metabolism in hematopoietic cells, with significant consequences on nuclear receptor-regulated pathways and atherosclerosis development.

Development of a new tandem mass spectrometry method for urine and amniotic fluid screening of oligosaccharidoses.

RATIONALE: The first step in the diagnosis of oligosaccharidoses is to evidence abnormal oligosaccharides excreted in urine, usually performed by the poorly sensitive but efficient thin layer chromatography (TLC) method. Developing a tandem mass spectrometry (MS/MS) technique could be of great interest to replace TLC. METHODS: Abnormal underivatized oligosaccharides have been recently studied using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, allowing the unambiguous identification of oligosaccharidoses. Based on this previous work, we developed an advantageous and efficient liquid chromatography (LC)/MS/MS method using a more common triple quadrupole tandem mass spectrometer for oligosaccharides analysis. RESULTS: Oligosaccharidoses (n = 97) and control (n = 240) urine samples were analysed. A specific pattern was obtained for each oligosaccharidosis using this method. In urine, it allows not only the identification of all the oligosaccharidoses previously identified by TLC (fucosidosis, alphamannosidosis, aspartylglucosaminuria, GM1 gangliosidosis, sialidosis, galactosialidosis and Schindler disease), but also extends the field of diagnosis to mucolipidosis type II, Sandhoff disease, and ß-mannosidosis. The same technique was applied to 16 amniotic fluid supernatants from oligosaccharidosis-affected foetuses (n = 16) compared with 37 unaffected. All the affected foetuses could be clearly identified: sialidosis (n = 3), galactosialidosis (n = 4), aspartylglucosaminuria (n = 1), mucolipidosis type II (n = 4) or GM1 gangliosidosis (n = 4). This technique can be applied to early prenatal diagnosis as well as to the oligosaccharidosis screening in the case of non-immune hydrops fetalis. CONCLUSIONS: The method is quick and easy to run, with an LC analysis time of 13 min per sample. The quantitative validation could not be obtained in the absence of a specific standard and of a labelled internal standard for each compound. Even if this LC/MS/MS method is only qualitative, it is very specific and much more sensitive than TLC. It allows the urinary screening of oligosaccharidoses, even mild or late-onset forms, and the screening of antenatal forms in amniotic fluid. Copyright © 2017 John Wiley & Sons, Ltd.

Fatty acid metabolism in macrophages: a target in cardio-metabolic diseases.

PURPOSE OF REVIEW: Recent studies have highlighted that macrophages dynamically and autonomously handle all the facets of fatty acid (FA) metabolism including FA oxidation and FA synthesis as well as the synthesis of monounsaturated FAs and long chain n-3 and n-6 polyunsaturated FAs. RECENT FINDINGS: Macrophage M2 polarization is associated with an increase of FA oxidation. However, whether increased FA oxidation simply correlates with or is required for M2 polarization needs to be further evaluated. Macrophage M1 polarization is associated with the activation of FA synthesis, which directly contributes to the inflammatory response and affects cholesterol homeostasis and neutral lipid accumulation. Finally, recent evidences suggest that macrophages are able to autonomously produce signaling monounsaturated FAs, such as palmitoleic acid (C16 : 1 n-7), and long chain n-3 and n-6 polyunsaturated FAs, such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. This pathway is regulated by liver X receptors and has significant consequences on inflammation and on the FA composition of atheroma plaques. SUMMARY: These studies shed new light on the tight relationship between FA metabolism, macrophage polarization, and M1/M2 macrophage functions. These processes may have major consequences for atherosclerosis pathogenesis as well as other metabolic disorders.

Extreme hyperferritinemia in the setting of acute myeloid leukaemia: a case report of hemophagocytic lymphohistiocytosis.

INTRODUCTION: Major hyperferritinemia is a rare feature in clinical laboratories associated with a wide variety of disorders, including hemophagocytic lymphohistiocytosis (HLH). The diagnosis of HLH is based on clinical and biological criteria, such as those proposed by the Histiocyte Society. However, several of these criteria are not relevant in the specific setting of hematologic malignancies. MATERIALS AND METHODS: A 69-year-old male was treated for an acute myeloid leukaemia. On day 15 after the start of chemotherapy, he developed severe sepsis with high fever, low blood pressure and hepatosplenomegaly. RESULTS: Blood tests were marked by extreme hyperferritinemia (191,000 µg/L, reference range: 26-388 µg/L) with increased C-reactive protein (87.0 mg/L) and procalcitonin (1.94 µg/L) and aspartate aminotransferase (499 U/L 37 °C) in the setting of chemotherapy-induced aplasia. This unusual extreme ferritinemia led to suspect HLH triggered by an invasive infection. Under intensive treatment, the clinical status improved and ferritin levels significantly decreased. CONCLUSIONS: The diagnosis of HLH is usually based on clinical and biological criteria, mainly fever, splenomegaly, cytopenias, hypertriglyceridemia, hypofibrinogenemia, hemophagocytosis and hyperferritinemia. In this patient, the diagnosis of HLH was challenging because several criteria, such as hypertriglyceridemia, hemophagocytosis and hypofibrinogenemia, were absent. In addition, some criteria of HLH are not relevant in the setting of hematologic malignancy, in which fever, splenomegaly, cytopenias and elevated lactate dehydrogenase are commonly observed independently of HLH. This unusual case of extremely high ferritinemia emphasizes the important weight of the ferritin level for the diagnosis of HLH in adult patients in the setting of hematologic malignancies.

Specific enrichment of 2-arachidonoyl-lysophosphatidylcholine in carotid atheroma plaque from type 2 diabetic patients.

BACKGROUND AND AIMS: Diabetic patients are at high risk of stroke and coronary artery disease. Recent data suggest that arachidonic acid metabolism is altered in diabetic conditions and that these alterations contribute to accelerated atherosclerosis. Little is known about how these alterations affect the metabolism and the proportions of different lipid species within the atherosclerotic plaque. The aim of our study was to perform a targeted lipidomic analysis of human atherosclerotic lesions, with a specific focus on PUFA-containing lipid species, to reveal differences in the fatty-acid composition of plaque in diabetic patients compared with non-diabetic controls. METHODS: Carotid atheroma plaque samples were obtained from 31 diabetic and 48 non-diabetic patients undergoing carotid endarterectomy. Targeted lipidomic analysis was then performed to determine the fatty acid composition of major glycerophospholipids and cholesteryl ester species by liquid chromatography-tandem mass spectrometry. RESULTS: Atheroma plaques from diabetic patients were significantly enriched with 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) (2.3 ± 0.8% Vs. 1.8 ± 0.6% p = 0.0002). Multivariable logistic regression showed that an increased 2-AA-LPC level was independently associated with diabetes. Finally, a positive relationship was found between 2-AA-LPC and HbA1c levels. Interestingly, endothelial lipase and calcium independent PLA2 gamma which could account for the production of 2-AA-LPC were detected in carotid plaques by immunohistochemistry. CONCLUSIONS: 2-AA-LPC stands at the crossroads of major metabolic pathways that lead to the synthesis of bioactive molecules such as AA-derived eicosanoids, 2-AA-lysophosphatidic acid and 2-AA-glycerol. 2-AA-LPC therefore appears to be a promising molecule to investigate in the context of diabetes.

Are Adiponectin and Leptin Good Predictors of Surgical Infection after Colorectal Surgery? A Prospective Study.

BACKGROUND: Infections are the most frequent complication after colorectal surgery. It has been suggested that adipose tissue metabolism could be related to the risk of post-operative infection, but this could be partially related to the body-mass index. The aim of this study was to look for a relation between adipocytokine levels and the risk of post-operative infection and its type. METHODS: This prospective cohort study was conducted between March 2013 and March 2014 in two French teaching hospitals. Pre-operative plasma levels of adiponectin and leptin were measured in consecutive patients undergoing elective colorectal surgery. All infections in the 30 d following surgery were recorded. RESULTS: Among the 142 patients included, 29 (20.4%) presented a post-operative infection: 26 surgical site infections and three extra-abdominal infections. Adiponectin and leptin levels correlated weakly but substantially with the body mass index (rspearman=-0.26 and +0.31, respectively). While there was no substantial difference between patients with and those without post-operative infection for adiponectin, median pre-operative leptin was substantially greater in patients with post-operative infection (8.67 vs. 4.37 ng/mL, p=0.003). A substantial interaction was found between leptin and cancer. In patients operated on for cancer, the area under the receiver-operating characteristic (ROC) curve was lower than in patients with benign diseases (0.597 vs. 0.858, p=0.011). Similar results were observed for intra-abdominal infection and surgical site infection. CONCLUSION: Patients with greater levels of leptin before colorectal surgery have an increased risk of post-operative surgical infection. This effect is stronger in patients without cancer. Adiponectin levels are not related to the risk of infection in Western patients.

Liver X receptor activation promotes polyunsaturated fatty acid synthesis in macrophages: relevance in the context of atherosclerosis.

OBJECTIVE: Liver X receptors (LXRs) modulate cholesterol and fatty acid homeostasis as well as inflammation. This study aims to decipher the role of LXRs in the regulation of polyunsaturated fatty acid (PUFA) synthesis in macrophages in the context of atherosclerosis. APPROACH AND RESULTS: Transcriptomic analysis in human monocytes and macrophages was used to identify putative LXR target genes among enzymes involved in PUFA biosynthesis. In parallel, the consequences of LXR activation or LXR invalidation on PUFA synthesis and distribution were determined. Finally, we investigated the impact of LXR activation on PUFA metabolism in vivo in apolipoprotein E-deficient mice. mRNA levels of acyl-CoA synthase long-chain family member 3, fatty acid desaturases 1 and 2, and fatty acid elongase 5 were significantly increased in human macrophages after LXR agonist treatment, involving both direct and sterol responsive element binding protein-1-dependent mechanisms. Subsequently, pharmacological LXR agonist increased long chain PUFA synthesis and enhanced arachidonic acid content in the phospholipids of human macrophages. Increased fatty acid desaturases 1 and 2 and acyl-CoA synthase long-chain family member 3 mRNA levels as well as increased arachidonic acid to linoleic acid and docosahexaenoic acid to eicosapentaenoic acid ratios were also found in atheroma plaque and peritoneal foam cells from LXR agonist-treated mice. By contrast, murine LXR-deficient macrophages displayed reduced expression of fatty acid elongase 5, acyl-CoA synthase long-chain family member 3 and fatty acid desaturases 1, as well as decreased cellular levels of docosahexaenoic acid and arachidonic acid. CONCLUSIONS: Our results indicate that LXR activation triggers PUFA synthesis in macrophages, which results in significant alterations in the macrophage lipid composition. Moreover, we demonstrate here that LXR agonist treatment modulates PUFA metabolism in atherosclerotic arteries.